Significance: Salvinorin A, a naturally occurring neoclaridane diterpine and the main active ingredient of the 'magic mint' hallucinogenic plant Salvia divinorum, is a potent and selective k-opioid receptor agonist (Roth et al., Proc Natl Acad Sci USA, 2002). Salvia divinorum and Salvinorin A thus represent a novel class of abused drugs (Sheffler and Roth, Trends Pharmacol Sci, in press) which selectively target the kappa-opioid receptor. The discovery that a diterpine is a selective peptide receptor agonist is unique and unprecedented. Therefore, the elucidation of the structural features responsible for Salvinorin A's actions at k-opioid receptors will illuminate novel approaches for peptide drug design. Investigation of the interaction of Salvinorin A with the k-opioid receptor may also provide a case study representing an innovative strategy for the design of drugs with exceptionally high selectivity. Thus, non-amine ligands, such as salvinorin A, have a potential for nearly absolute selectivity. To accomplish this overall goal we will test the following two hypotheses and 5 specific aims: Hypothesis #1: Salvinorin A interacts with the k-opioid receptor in a unique manner. Specific aim #1: To determine the structural features of the k-opioid receptor essential for binding Salvinorin A via a combination of site-directed mutagenesis and molecular modeling. Specific aim #2: To determine the structural features of the k-opioid receptor essential for the agonist actions of Salvinorin A at the KOR via a combination of site-directed mutagenesis and molecular modeling approaches. Specific aim #3: To compare the mode of binding and activation of the KOR by Salvinorin A with selected alkaloid agonists. Hypothesis #2: The 2-methoxycarbonyl position of Salvinorin A is essential for binding to and activation of the k-opioid receptor. Specific aim #4: To compare the binding and functional properties of a series of naturally occurring and synthetic 2-substituted Saivinorin A derivatives. These studies are likely to clarify how Salvinorin A and related drugs of abuse mediate their actions at the molecular and cellular levels and will lead to treatments for the side-effects related to Salvinorin A abuse.